In this specification, a number of documents are cited. The disclosure of these documents, including manufacturer's manuals and patent applications or patents, is herewith incorporated by reference in its entirety.
GPCRs represent the largest family of cell surface receptors with an estimated number of up to 1000 genes within the human genome characterized by a seven-transmembrane configuration as their main feature. (Bockaert and Pin, 1999; Pierce et al., 2002). GPCRs are activated by a multitude of different ligands, including peptides, proteins, lipids, small molecules, ions or even photons. Activated GPCRs alter their conformation allowing it to catalyze the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on the α-subunit of a heterotrimeric g-protein coupled to the GPCR. Heterotrimeric G-proteins composed of one out of 18 different α-subunits, one out of 5 different β-subunits and one out of 11 different γ-subunits are usually classified by the nature of their α-subunit and generally grouped into four main classes: Gs, which activates adenylyl cyclase; Gi, which inhibits adenylyl cyclase; Gq, which activates phospholipase C; and G12/13 with heterologous functions. In addition to the α-subunit dependent signaling the β/γ-subunits can function as signaling molecules on their own. GPCR dependent signaling becomes even more complex if it is considered that these receptors can exist as homo-oligomeric or hetero-oligomeric complexes. (George et al., 2002; Milligan et al., 2003; Salahpour et al., 2000). Hence, it is not surprising that GPCRs are responsible for the regulation of a wide variety of different physiological processes.
Recently the role of GPCRs in human senses like vision, olfaction and taste has been subject of intensified investigations. While the participation of the GPCR rhodopsin in visual sensing is one of the most comprehensively examined g-protein coupled receptor signaling examples of the last 30 years (Maeda et al., 2003), the role of GPCRs in olfaction and bitter taste as well as sweet taste was discovered in the 1990ies. (Buck and Axel, 1991; Firestein, 2001; Lindemann, 1996b; Lindemann, 2001).
The discovery of GPCR signaling in taste perception is closely connected to the discovery of tastant specific signaling in vertebrate taste cells. In electrophysical and biochemical studies it was apparent that tastant derived signaling resulted in typical GPCR dependent second messenger induction, e.g. cyclic nucleosides (cAMP, cGMP), inositol tri-phosphate (IP3) or calcium. (Kinnamon und Cummings, 1992; Kinnamon und Margolskee, 1996; Lindemann, 1996a). The participation of GPCRs in taste perception was further approved by the finding of the g-protein gustducin specifically expressed in vertebrate taste cells. (McLaughlin et al., 1992; Wong et al., 1996). On the other hand it was known from genetic mouse studies that the ability to sense sweet taste of e.g. saccharin was linked to the so called sac locus on mouse chromosome 4. (Bachmanov et al., 2001; Lush, 1989; Lush et al., 1995). Based on these data it was obvious to search for GPCR sequence tags in taste cell derived subtracted cDNA libraries or by performing genomic sequence scanning to further narrow down the mouse sac locus for the identification of GPCR analogs as putative taste receptors. These two approaches led to the rat, mouse and human receptor DNA sequences for the taste GPCRs T1R1 and T1R2 (Hoon et al., 1999; Hoon and Ryba, 1997) as well as T1R3. (Kitagawa et al., 2001; Li et al., 2001; Max et al., 2001; Montmayeur et al., 2001; Sainz et al., 2001). Homology alignments revealed that these taste receptors like the homodimeric metabotrophic glutamate receptor (mGluR), the heterodimeric γ-aminobutyric acid type B receptor (GABABR) and homodimeric extracellular calcium receptors are members of the small family of class C GPCRs. As a common characteristic most of the class C receptors exhibit a large extracellular amino terminal domain composed of a so called venus flytrap module (VFTM) and a cysteine rich domain (CRD) that connects the VFTM to the heptahelical domain. (Pin et al., 2003). Besides that homo- as well as hetero-oligomerisation was described for several of these class C receptors. (Bai et al., 1998; Kaupmann et al., 1998; Kunishima et al., 2000; White et al., 1998). Consequently, the characteristic feature of GPCR oligomerisation of class C receptors was tested for the putative sweet taste receptors T1R1, T1R2 and T1R3.
By recombinant heterologous expression in eucaryotic cell systems a functional expression and tastant specific activation of an artificially linked G-Protein dependent signaling cascade was demonstrated by calcium imaging. T1R receptors assemble to build up functional taste receptors. As a result of several investigations it was shown that the heterodimeric T1R1/T1R3 functions as a glutamate (umami) and L-amino acid receptor whereas the heterodimeric T1R2/T1R3 functions as a high affinity sugar and artificial sweetener receptor. Particularly, heterodimeric co-expression of T1R1 and T1R3 results in taste receptors that respond to umami taste and monosodium glutamate stimuli whereas heterodimeric co-expression of T1R2 and T1R3 results in taste receptors that respond to sweet stimuli like diverse sugars (e.g. glucose and sucrose), artificial sweetener (e.g. acesulfam K, cyclamat, saccharin) and sweet proteins like monellin, thaumatin, brazzein (Li et al., 2002; Nelson et al., 2002; Nelson et al., 2001; Zhao et al., 2002). A similar chronicle could be generated for the identification of GPCRs for the perception of bitter taste with the exception that so far no homo- or oligomerisation has been reported for these so called T2R-GPCRs. (Meyerhof et al., 2005).
The above discussed identification of genes coding for receptors responsible e.g. for taste perception, together with cloning said genes into appropriate vectors for the expression of said proteins in eukaryotic cells and the transformation of said cells with said vectors raised the expectation that screening systems and/or screening methods for GPCR modulators, i.e. agonists and antagonists of the above detailed receptors should be easy to be developed within a reasonable time.
This is reflected by a huge and still growing number of patent applications in this field.
The cloning of T1R1 is disclosed in different patent applications, e.g. in WO 03/025137; in WO 00/06952 (wherein it is designated GPCR-B3) US020040191862A1 and WO2005/033125.
The cloning of T1R2 is disclosed in patent applications WO 03/025137, US020040191862A1 and US020030040045A1
The cloning of T1R3 is disclosed in patent applications WO 03/025137, WO 03/025137, US020040191862A1 and US020030040045A1
A system for the expression of said proteins in eukaryotic cells is disclosed in patent applications WO 03/025137, WO 00/06952, US20040191862A1, WO2004069191 and US20030040045A1.
A screening system for putative taste modulators is disclosed in patent applications WO 00/06952, WO2004069191 and US20030040045A1.
Yet, nothing is to be told about the successful identification of new modulators, e.g. new artificial taste modulators such as new sweeteners utilizing such screening methods/systems.
The ongoing debate on obesity in developed countries and the growing health consciousness of consumers lead to an increasing demand of food and beverages with significant calorie reduction compared to products fully sweetened with carbohydrates such as sucrose, glucose, fructose or syrups such as HFCS 55 or 42. As the consumer usually is not willing to compromise on taste products should have similar sweetness intensity and taste quality as products regularly sweetened with these carbohydrates.
High intensity sweeteners are substances, which have no or virtually no calories and a sweetness potency several times higher than sugar. High intensity sweeteners or blends of high intensity sweeteners are used in food and beverages to achieve a sweet taste without adding calories to the products.
Most commonly used high intensity sweeteners are not from natural origin; They were discovered accidentally and are chemically synthesized. Most of them have a widespread approval in a large number of countries. Examples are substances such as acesulfame K, alitame, aspartame, cyclamate, neohesperidine dihydrochalcone, neotame, saccharin, and sucralose.
However, no high-intensity sweetener matches the taste profile of sugar completely. They differ in characteristics such as sweetness profile, side taste and off-taste characteristics. Proper blending of different high intensity sweeteners is known to overcome part of the taste limitations of single high-intensity sweeteners. But even if a more sugar-like sweetness profile is achieved in products with high-intensity sweeteners only, they still can be distinguished sensorically from their counterparts with just sugar or other carbohydrates by lack of mouthfeel and reduced flavour characteristics. Therefore a need exists for new high-intensity sweeteners which offer either alone or in blends with existing sweeteners sweetness profiles and flavour characteristics much closer to sugar than the existing products can offer.
Besides calorie reduction many of today's consumers are seeking for food and beverage products either without artificial additives or even being fully organic. Theoretically natural high-intensity sweeteners could fulfil this demand. A number of natural high-intensity sweeteners were discovered throughout past years such as stevioside, rebaudioside, brazzein, thaumatin, mogroside, glycyrrhizin, monatin, abrusoside, monellin, phyllodulcin and others. These are substances which naturally occur in different plants and can be obtained by selective extraction measures. Besides very limited approvals and in some cases difficulties to extract products on an industrial scale none of these products can claim to offer a sugar-like taste. In fact, all of these substances show a sweetness with a far slower onset than sucrose and a very lingering sweetness. Most of these products have strong side-taste and aftertaste characteristics such as bitter, mentholic or liquorice notes or show even strong cooling or numbing sensations. Therefore some of these products, e.g. thaumatin, can be rather regarded as being flavour enhancer than sweetener. Blending of two or more of these substances can not overcome these taste limitations. Therefore in the area of natural sweetener the need for new high-intensity sweeteners with a taste profile closer to sugar is even stronger than in the case of artificial sweeteners (O'Brien Nabors, 2001; Leatherhead Food R A, 2000; Grenby, 1996; von Rymon Lipinski und Schiweck, 1991).
Therefore, there still exists a need in the art to identify and isolate new substances which may be used as modulators of taste perception, e.g. as sweeteners.
Notwithstanding the above, because of the high importance of these GPCRs in vivo, and the many different functions associated with said receptors, it has to be assumed that many of the modulators of GPCRs that might be identified by the method of the present invention may be of practical value.
Therefore, the availability of a simple and reliable screening system for modulators of said receptors would be of big importance.
In multicistronic expression vectors the coding sequences of different proteins are under the control of only one promoter and the different cistrons are connected via virus derived internal ribosomal entry sites (IRES) or cap independent translation enhancer (CITE). IRES or CITE elements confer a translation initiation independent from the otherwise necessary 5′-end of a messenger RNA, which is recognized by the eucaryotic ribosomes to start their scanning process for the first accessible translational start codon. (Fux et al., 2004; Hellen and Sarnow, 2001). So far multicistronic expression vectors have been described basically as dicistronic expression units for the coupled expression of a gene of interest linked via an cap-independent translation initiation site to a resistance marker (confering resistance to e.g. hygromycin, zeocin, neomycin) enabling selection of stable cell lines for heterologous mammalian expression studies. For this approach IRES or CITE dependent expression vectors are commercially available.
Reports on genuine multicistronic expression studies in mammalian systems with descriptions of tri-cistronic or even quadro-cistronic heterologous expression studies are rare and for the most part intended to improve inducible protein expression e.g. for gene therapy applications. However in this pioneering work multicistronic expressions have been mostly performed with small and soluble proteins like reporter genes (green fluorescent protein, yellow fluorescent protein, red fluorescent proteins, secreted alkaline phosphatase, secreted amylase) or engineered transactivators e.g. for macrolide- or streptogramin dependent expression or selection markers. Although these studies are aimed at potential therapeutic protein expression in gene therapy applications, only few genes with a therapeutic potential are mentioned within this studies (e.g. vascular endothelial growth factor (VEGF); the oncoprotein bcl-2). (Fussenegger et al., 1998; Hartenbach and Fussenegger, 2005; Kramer et al., 2003; Moser et al., 2000; Weber et al., 2005).
Concerning the expression of taste receptors there is one report on dicistronic expression of mouse taste receptors (mT2R8/5; mT1R3) each fused to green fluorescent protein and linked via an IRES element to red fluorescent protein. This approach was applied to trace and localize the expression pattern of taste receptors in neurons (Sugita and Shiba, 2005).